PROJECT SUMMARY Client proteins of the secretory pathway fold to their native shapes in the endoplasmic reticulum (ER) through reactions catalyzed by chaperones and other ER protein-modifying enzymes. Under high secretory demand, these activities are overwhelmed, causing unfolded proteins to accumulate. If uncorrected, such ?ER stress? increases the risk of cell degeneration and death. Photoreceptors, specialized neurons in the retina responsible for phototransduction, have one of the highest secretory burdens of any human cell, making them particularly susceptible to ER stress. Recent evidence has implicated pathogenic ER stress as a potential cause of retinitis pigmentosa (RP), a blinding disease marked by the progressive loss of photoreceptors, especially in cases due to mutations in rhodopsin that prevents its proper folding. Accumulation of unfolded proteins in the ER triggers signaling pathways called the unfolded protein response (UPR). Under remediable levels of ER stress, the adaptive UPR (A-UPR) activates transcriptional and translational changes that restore homeostasis. However, under irremediably high ER stress, these adaptive measures fail and the signaling pathways instead trigger programmed cell death?referred to as the terminal UPR (T-UPR). We discovered that the ER transmembrane protein IRE1?, a bifunctional kinase/endoribonuclease (RNase), converts an A-UPR to a T-UPR. During rectifiable ER stress, IRE1? transiently trans-autophosphorylates, causing its RNase to trigger the A-UPR through frame-shift splicing of the mRNA encoding XBP1 transcription factor. But under high/chronic ER stress, IRE1?'s kinase becomes hyperphosphorylated, causing RNase hyperactivation that leads to massive degradation of ER-localized mRNA and T-UPR events including: (1) loss of differentiated cell identity, (2) local sterile inflammation, and (3) programmed cell death through pyroptosis and apoptosis. We hypothesize that an IRE1?-induced switch from an A-UPR to a T-UPR contributes to ER stress-induced photoreceptor loss. Our overall goal for this R01 is threefold: (1) define the role of IRE1? signaling on normal photoreceptor health; (2) elucidate key underlying molecular mechanisms through which IRE1? converts an A- UPR to a T-UPR in photoreceptors; and (3) target IRE1? in photoreceptors using our recently developed kinase inhibitors for long-term prevention of retinal degeneration. Our research project, to be driven by three labs with complementary and synergistic skills as well as experienced collaborators, promises to provide powerful mechanistic insights into the role of the UPR in photoreceptor health and degeneration, and to establish whether the UPR can be successfully drugged to prevent photoreceptor loss in RP.